Comparative Analysis of the Mitochondrial Genomes of Chloropidae and Their Implications for the Phylogeny of the Family

Chloropidae, commonly known as grass flies, represent the most taxonomically diverse family of Diptera Carnoidea, comprising over 3000 described species worldwide. Previous phylogenetic studies of this family have predominantly relied on morphological characters, with mitochondrial genomes being reported in a few species. This study presents 11 newly sequenced mitochondrial genomes (10 Chloropidae and 1 Milichiidae) and provides the first comprehensive comparative analysis of mitochondrial genomes for Chloropidae. Apart from 37 standard genes and the control region, three conserved intergenic sequences across Diptera Cyclorrhapha were identified in all available chloropid mitochondrial genomes. Evolutionary rates within Chloropidae exhibit significant variation across subfamilies, with Chloropinae displaying higher rates than the other three subfamilies. Phylogenetic relationships based on mitochondrial genomes were inferred using maximum likelihood and Bayesian methods. The monophyly of Chloropidae and all four subfamilies is consistently strongly supported, while subfamily relationships within Chloropidae remain poorly resolved, possibly due to rapid evolution.


Introduction
Chloropidae, commonly known as grass flies, is the most taxonomically diverse family of Carnoidea.It currently comprises 203 described genera and over 3000 known extant species worldwide [1,2].Their bodies are usually mostly black or primarily yellow with black to brown stripes, and they have diverse body shapes, from short and broad to greatly elongated, with their total lengths ranging from 1.0 to 9.5 mm in size [3,4].The origins of this family coincide with the schizophoran radiation during the early Tertiary [5], and the reported fossils have provided a glimpse into their diversity in the Eocene and Oligocene [6].
The subfamily-level classification of Chloropidae has been widely discussed over recent decades (Figure 1).Members of Chloropidae are currently assigned to three or four subfamilies.Andersson [17] established a classification into three subfamilies-Siphonellopsinae, Chloropinae, and Oscinellinae-and described the tribe Rhodesiellini in the Oscinellinae.This division was accepted by Kanmiya [3], Sabrosky [18], Wheeler [19], Ismay and Nartshuk [20], Mlynarek and Wheeler [21], and Ismay et al. [4].Nartshuk [22,23] treated Rhodesiellinae as a subfamily, which was followed by Cherian [24], Nartshuk [1], Nartshuk and Andersson [15], and Riccardi and Amorim [2], resulting in the recognition of Chloropidae comprising four subfamilies.Andersson [12,25] initially intended to reconstruct the phylogeny of the family.Due to high diversification and relatively recent evolution of chloropids, Andersson identified a considerable challenge in conducting a cladistic analysis of the family based on morphology.In his study, Andersson [25] analyzed general phylogenetic relationships within the family based on morphological data and concluded that Siphonellopsinae is the 'basal subfamily' (Figure 1).This is supported by most studies based on morphological data [2,3,23,26].The relationships between Chloropinae, Oscinellinae, and Rhodesiellinae are, however, confusing.Nartshuk and Andersson [15] suggested either Chloropinae + (Oscinellinae + Rhodesiellinae) or Oscinellinae + (Chloropinae + Rhodesiellinae).Bazyar [27] suggested Chloropinae as the sister group of Siphonellopsinae + (Oscinellinae + Rhodesiellinae) (Figure 1).However, the Rhodesiellinae is poorly defined in his study, and the set of genera included in Rhodesiellinae corresponds to an early branching group of genera at the base of the Oscinellinae [27].
The dipteran mitochondrial genomes are relatively conservative in size and structure and rarely occur in gene rearrangement.Dipterans follow similar codon usage and nucleotide biases which are possibly influenced by mutational and selection pressures [28].The mitochondrial genome phylogeny can largely reduce random phylogenetic errors arising from short genes or the high or low conservatism of individual genes [29].Mitochondrial genomes have been widely used for phylogenetic inference for various taxonomic lineages of dipterans [30][31][32] because of their typically uniparental inheritance, lack of in-trons, high copy numbers, relatively simple structure, conserved gene composition, and rapid evolutionary rate [30].However, limited attention has been given to individual chloropid mitochondrial genomes, with studies focusing primarily on Anatrichus pygmaeus [33], Chlorops oryzae [34], and Dicraeus orientalis [35].This lack of data hinders a comprehensive phylogenetic analysis of Chloropidae.To elucidate the relationships among chloropid subfamilies, it is crucial and pressing to generate more mitochondrial genomes across the family and undertake comparative and phylogenetic analyses.
In this study, the mitochondrial genomes of 10 Chloropidae that cover all four subfamilies were newly generated and annotated, along with one Milichiidae outgroup.We integrated previously reported chloropid mitochondrial genomes; analyzed their genomic structures, nucleotide compositions, and substitutional and evolutionary rates; and reconstructed the phylogeny of the family.We aimed to (1) assess the validity of Rhodesiellinae and (2) to elucidate the subfamily-level relationships within Chloropidae.

General Features and Genome Organization
The mitochondrial genomes of ten chloropid species were successfully sequenced, resulting in five complete genomes: Apotropina sp.1, Elachiptera insignis, Rhodesiella sp., Rhodesiella elegantula, and Thaumatomyia glabra.These mitochondrial genomes are typically double-stranded circular molecules containing 37 genes (13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes) and a control region (CR).Together with three previously available data, eight complete chloropid mitochondrial genomes were then utilized for our comparative analysis.The length of chloropid mitochondrial genomes ranged from 16,033 bp (Apotropina sp. 1) to 17,313 bp (Chlorops oryzae).The lengths of PCGs, tRNAs, and rRNAs for Chloropidae are nearly identical.However, significant size variation was observed in the control region (Figure 2).The majority strand (J-strand) encodes 23 genes, including 9 PCGs and 14 tRNAs, while the remaining 14 genes (4 PCGs, 8 tRNAs, and 2 rRNAs) are transcribed from the minority strand (N-strand).The gene order in chloropid mitochondrial genomes aligns with that of previously published mitochondrial genomes of Schizophora, demonstrating high conservation in Brachycera.
from short genes or the high or low conservatism of individual genes [29].Mi genomes have been widely used for phylogenetic inference for various taxonom of dipterans [30][31][32] because of their typically uniparental inheritance, lack of i copy numbers, relatively simple structure, conserved gene composition, and r tionary rate [30].However, limited attention has been given to individual chlo chondrial genomes, with studies focusing primarily on Anatrichus pygmaeus [3 oryzae [34], and Dicraeus orientalis [35].This lack of data hinders a comprehen genetic analysis of Chloropidae.To elucidate the relationships among chlorop ilies, it is crucial and pressing to generate more mitochondrial genomes acros and undertake comparative and phylogenetic analyses.
In this study, the mitochondrial genomes of 10 Chloropidae that cover a families were newly generated and annotated, along with one Milichiidae ou integrated previously reported chloropid mitochondrial genomes; analyze nomic structures, nucleotide compositions, and substitutional and evolutionar reconstructed the phylogeny of the family.We aimed to (1) assess the validity ellinae and (2) to elucidate the subfamily-level relationships within Chloropid

General Features and Genome Organization
The mitochondrial genomes of ten chloropid species were successfully resulting in five complete genomes: Apotropina sp.1, Elachiptera insignis, Rh Rhodesiella elegantula, and Thaumatomyia glabra.These mitochondrial genomes a double-stranded circular molecules containing 37 genes (13 protein-coding ge genes, and 22 tRNA genes) and a control region (CR).Together with three available data, eight complete chloropid mitochondrial genomes were then our comparative analysis.The length of chloropid mitochondrial genomes r 16,033 bp (Apotropina sp. 1) to 17,313 bp (Chlorops oryzae).The lengths of PC and rRNAs for Chloropidae are nearly identical.However, significant size va observed in the control region (Figure 2).The majority strand (J-strand) encod including 9 PCGs and 14 tRNAs, while the remaining 14 genes (4 PCGs, 8 tR rRNAs) are transcribed from the minority strand (N-strand).The gene order i mitochondrial genomes aligns with that of previously published mitochondr of Schizophora, demonstrating high conservation in Brachycera.

Base Composition
The Chloropidae mitochondrial genomes exhibit a noticeable A+T bias, ranging from 78.5% (Thaumatomyia glabra) to 80.9% (Elachiptera insignis).In the PCGs, the base composition of each codon shows that the third codon positions have much higher A + T content compared to the first and second codon positions.All Chloropidae mitochondrial genomes show a positive AT-Skew and a negative GC-Skew (Figure 3).
The Chloropidae mitochondrial genomes exhibit a noticeable A+T bias, ranging from 78.5% (Thaumatomyia glabra) to 80.9% (Elachiptera insignis).In the PCGs, the base composition of each codon shows that the third codon positions have much higher A + T content compared to the first and second codon positions.All Chloropidae mitochondrial genomes show a positive AT-Skew and a negative GC-Skew (Figure 3).

Protein-Coding Genes, Codon Usage, and Evolutionary Rate Analysis
The total length of the 13 PCGs in the 14 Chloropidae flies ranges from 11,170 bp (Dicraeus orientalis) to 11,252 bp (Pachylophus sp.).ATP8 is the shortest gene, while the largest gene is ND5.Most of the 13 PCGs start with the standard start codon ATN; however, in some cases, this pattern does not hold.For instance, COI of Cetema sp., Chlorops oryzae, and Meromyza saltatrix starts with ACG, and that of Anatrichus pygmaeus, Apotropina sp. 1, Apotropina sp. 2, Cadrema minor, Dicraeus orientalis, Elachiptera insignis, Oscinella pusilla, Rhodesiella sp., and Rhodesiella elegantula starts with TCG.Additionally, the COII of Rhodesiella sp.starts with GTG; the ND1 of Apotropina sp. 2, Cadrema minor, Elachiptera insignis, Meromyza saltatrix, Oscinella pusilla, Rhodesiella sp., Rhodesiella elegantula, and Thaumatomyia glabra starts with TTG; and the start codon for ND5 is GTT in Cetema sp. and CTC in Meromyza saltatrix.All PCGs end with TAA, TAG, or truncated termination codons such as TA or single T. The relative synonymous codon usage (RSCU) of mitogenomes across all subfamilies was calculated (Figure S1).Ser2 and Leu2 are the two most frequently utilized amino acids in Chloropidae.
To better comprehend the evolutionary rate of PCGs, we analyzed the mean ratios (ω) of non-synonymous to synonymous substitutions of the 13 PCGs to represent the selection pressures.Most of the ω values of PCGs are less than 1, except for the ATP8 of some species (Apotropina sp.2: 1.10, Cadrema minor: 1.11, Cetema sp.1: 1.46, and Pachylophus sp.1: 1.20), which may be due to its sequence being too short (Figure S2).This result suggested that these protein-coding genes are evolving under a purifying selection.Within these PCGs, on average, ATP8 (with ω = 0.89) demonstrates a relatively high value, suggesting it has undergone relaxed selection.By contrast, COI possesses the lowest Ka/Ks ratio (with ω = 0.07) and exhibits a strong purifying selection.

Protein-Coding Genes, Codon Usage, and Evolutionary Rate Analysis
The total length of the 13 PCGs in the 14 Chloropidae flies ranges from 11,170 bp (Dicraeus orientalis) to 11,252 bp (Pachylophus sp.).ATP8 is the shortest gene, while the largest gene is ND5.Most of the 13 PCGs start with the standard start codon ATN; however, in some cases, this pattern does not hold.For instance, COI of Cetema sp., Chlorops oryzae, and Meromyza saltatrix starts with ACG, and that of Anatrichus pygmaeus, Apotropina sp. 1, Apotropina sp. 2, Cadrema minor, Dicraeus orientalis, Elachiptera insignis, Oscinella pusilla, Rhodesiella sp., and Rhodesiella elegantula starts with TCG.Additionally, the COII of Rhodesiella sp.starts with GTG; the ND1 of Apotropina sp. 2, Cadrema minor, Elachiptera insignis, Meromyza saltatrix, Oscinella pusilla, Rhodesiella sp., Rhodesiella elegantula, and Thaumatomyia glabra starts with TTG; and the start codon for ND5 is GTT in Cetema sp. and CTC in Meromyza saltatrix.All PCGs end with TAA, TAG, or truncated termination codons such as TA or single T. The relative synonymous codon usage (RSCU) of mitogenomes across all subfamilies was calculated (Figure S1).Ser2 and Leu2 are the two most frequently utilized amino acids in Chloropidae.
To better comprehend the evolutionary rate of PCGs, we analyzed the mean ratios (ω) of non-synonymous to synonymous substitutions of the 13 PCGs to represent the selection pressures.Most of the ω values of PCGs are less than 1, except for the ATP8 of some species (Apotropina sp.2: 1.10, Cadrema minor: 1.11, Cetema sp.1: 1.46, and Pachylophus sp.1: 1.20), which may be due to its sequence being too short (Figure S2).This result suggested that these protein-coding genes are evolving under a purifying selection.Within these PCGs, on average, ATP8 (with ω = 0.89) demonstrates a relatively high value, suggesting it has undergone relaxed selection.By contrast, COI possesses the lowest Ka/Ks ratio (with ω = 0.07) and exhibits a strong purifying selection.
The subfamily Chloropinae exhibits a higher substitution value than Oscinellinae, Siphonellopsinae, and Rhodesiellinae (Figure 4).High substitution accumulation and faster changes in the genetic sequence suggest that the species has undergone significant genetic changes in a relatively short time, indicating potentially rapid diversification [36].
In contrast to the other three subfamilies, Chloropinae exhibits higher base substitution rates and genetic distances, implying that they may have undergone rapid evolution.
The subfamily Chloropinae exhibits a higher substitution value than Oscine Siphonellopsinae, and Rhodesiellinae (Figure 4).High substitution accumulatio faster changes in the genetic sequence suggest that the species has undergone sign genetic changes in a relatively short time, indicating potentially rapid diversificatio In contrast to the other three subfamilies, Chloropinae exhibits higher base subst rates and genetic distances, implying that they may have undergone rapid evolutio

Intergenic Sequences
Previous studies on mitochondrial genomes of Cyclorrhapha have identified conserved intergenic spacers: 18 bp between ND1 and tRNA Ser (UCN) , 18 bp between tR and tRNA Phe , and 15 bp between tRNA His and ND5 [31,32].All three of these con intergenic spacers were detected in chloropid mitochondrial genomes.Specifical spacer between tRNA Glu and tRNA Phe contains a 14 bp conserved sequence across examined chloropid flies; the spacer between ND1 and tRNA Ser (UCN) exhibits a 16 b served sequence, and the spacer between ND5 and tRNA His shows a 15 bp conserv quence.Conserved intergenic sequence blocks are presented in Figure 5.

Intergenic Sequences
Previous studies on mitochondrial genomes of Cyclorrhapha have identified three conserved intergenic spacers: 18 bp between ND1 and tRNA Ser (UCN) , 18 bp between tRNA Glu and tRNA Phe , and 15 bp between tRNA His and ND5 [31,32].All three of these conserved intergenic spacers were detected in chloropid mitochondrial genomes.Specifically, the spacer between tRNA Glu and tRNA Phe contains a 14 bp conserved sequence across all 14 examined chloropid flies; the spacer between ND1 and tRNA Ser (UCN) exhibits a 16 bp conserved sequence, and the spacer between ND5 and tRNA His shows a 15 bp conserved sequence.Conserved intergenic sequence blocks are presented in Figure 5.

Phylogenetic Analyses
The phylogenetic tree obtained from Bayesian and maximum likelihood (ML) analyses yielded similar topological structures across all four datasets, and the majority of the nodes are robustly supported.The monophyly of the Chloropidae, Rhodesiellinae, Siphonellopsinae, and Oseinellinae was consistently fully supported by all analyses (posterior probability = 1.00 for all datasets; ML bootstrap = 100 for all datasets), similar to the sister group relationship between Chloropidae and Milichiidae (Figure 6).This is consistent with previous phylogenetic studies [5,16].

Phylogenetic Analyses
The phylogenetic tree obtained from Bayesian and maximum likelih yses yielded similar topological structures across all four datasets, and the nodes are robustly supported.The monophyly of the Chloropidae, Rhodes nellopsinae, and Oseinellinae was consistently fully supported by all ana probability = 1.00 for all datasets; ML bootstrap = 100 for all datasets), sim group relationship between Chloropidae and Milichiidae (Figure 6).Th with previous phylogenetic studies [5,16].Relationships among four chloropid subfamilies were not robustly resolved, and three different topologies were generated based on eight phylogenetic estimations (Figure 6).Rhodesiellinae was weakly to fully supported as the sister group of remainders of the family by five analyses (NTR-ML, NTR-BI, NT123R-BI, AA-ML, and DegenR-BI) (Figure 6A).The remaining three analyses (NT123R-ML, DegenR-ML, and AA-BI) recovered Chloropinae as the earliest branching lineage of Chloropidae with fully supports (Figure 6B).
The monophyly of Siphonellopsinae + Oscinellinae was consistently supported in all analyses except AA-BI.This relationship was robustly supported by BI analyses with the degenerated nucleotide dataset and moderately supported by nucleotide datasets regardless of the partitioning schemes, whereas the ML method consistently provided weak supports (Figure 6A,B).The AA-BI analysis proposed Siphonellopsinae as the sister to Rhodesiellinae with weak supports (Figure 6C).Relationships among four chloropid subfamilies were not robustly resolved, and three different topologies were generated based on eight phylogenetic estimations (Figure 6).Rhodesiellinae was weakly to fully supported as the sister group of remainders of the family by five analyses (NTR-ML, NTR-BI, NT123R-BI, AA-ML, and DegenR-BI) (Figure 6A).The remaining three analyses (NT123R-ML, DegenR-ML, and AA-BI) recovered Chloropinae as the earliest branching lineage of Chloropidae with fully supports (Figure 6B).
The monophyly of Siphonellopsinae + Oscinellinae was consistently supported in all analyses except AA-BI.This relationship was robustly supported by BI analyses with the degenerated nucleotide dataset and moderately supported by nucleotide datasets regardless of the partitioning schemes, whereas the ML method consistently provided weak

Discussion
In this study, we analyzed 14 chloropid mitochondrial genomes, including 10 newly sequenced genomes from four subfamilies.The nucleotide composition is highly biased towards A + T, ranging from 78.5% to 80.9%, which is similar to that of other dipteran flies [30,31].Comparative analyses revealed that the chloropid mitogenomes are conserved in structure, which is consistent with all previously published mitochondrial genomes of Schizophora [31].The 13 PCGs present multiple types of starting codons: the standard start codon (e.g., ATN) and no standard start codon (e.g., ACG, TCG, GTG, TTG, GTT, CTC).This result has also been found in previous studies [31].The evolutionary rates of Chloropidae exhibit variation across subfamilies, with Chloropinae displaying a higher rate compared to the other three subfamilies, suggesting that Chloropinae may have undergone rapid evolution.
Three conserved intergenic sequences across Cyclorrhapha were found from all available chloropid mitochondrial genomes.Intergenic sequences serve multiple crucial roles in the genome.They contain regulatory elements such as promoters, enhancers, and transcription factor binding sites, which play a role in regulating gene expression [37].The non-coding region situated between ND1 and tRNA Ser (UCN) serves as the binding site for MtTERM, a highly conserved 7 bp motif across insects [38].MtTERM regulates the expression levels of the rRNA genes relative to the protein-coding genes [37,39].Furthermore, intergenic sequences are also important resources for the study of species evolution and phylogeny [40].
The subfamily-level relationships of Chloropidae have long been a topic of significant controversy across different phylogenetic analyses [3,15,[25][26][27].In the present study, maximum likelihood and Bayesian inference conducted on four datasets (NTR, NTR123, AA, and DegenR) indicated the monophyly of Chloropidae and all four subfamilies, which supports the previous hypothesis of monophyly [22,23,27].Initially proposed as a tribe of Oscinellinae [17], Rhodesiellinae was elevated to subfamily status by Nartshuk [22,23].However, the validity of Rhodesiellinae as a subfamily was doubted, as it was demonstrated to be paraphyletic in relation to the Oscinellinae based on morphological characters [27].Three analyses indicated Chloropinae as the earliest branching lineage of Chloropidae with strong supports, consistent with Bazya's findings based on morphological characters [27].Although some morphological studies also showed that Siphonellopsinae is a sister to the remaining Chloropidae [2,3,15], this relationship was never recovered in our analyses.
Our study provides the first comprehensive molecular-based subfamily-level phylogenetic estimates on Chloropidae.However, the subfamily relationships within Chloropidae remain unresolved.This could be due to our limited taxon sampling within the diverse group of Chloropidae and mitochondrial genomes may lack adequate phylogenetic signals for resolving these relationships.Future studies should consider incorporating a more comprehensive taxon and gene sampling to effectively address this issue.

Taxon Sampling and DNA Extraction
We newly sequenced mitochondrial genomes of 10 chloropid species representing all four subfamilies, as well as one Milichiidae outgroup (Table 1).Adult flies were collected by net-sweeping in the field and preserved in 100% ethanol at −20 • C before DNA extraction.Specimens were identified based on morphological characteristics by Xiaoyan Liu using keys in Kanmiya [3], Yang and Yang [41], and Nartshuk and Andersson [15].The genomic DNA was extracted from the thoracic muscle tissues of one specimen for each species using the DNeasy DNA Extraction kit (QIAGEN, Hilden, Germany).The remaining body parts of the sampled specimens were preserved as vouchers and deposited in the Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory at Huazhong Agricultural University, Wuhan, China.Specimen collection information and associated voucher numbers are listed in Tables 1 and S1.

Mitochondrial Genome Sequencing and Assembly
DNA samples were pooled for next-generation sequencing library construction following Gillett et al. [42].The library was sequenced on the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads, by Novogene CO., LTD.(Beijing China).The raw reads were filtered and trimmed using Fastp [43].De novo assemblies of high-quality reads were conducted using IDBA-UD [44] with a similarity threshold of 98% and minimum and maximum k values of 40 and 140 bp, respectively.The bait sequence COI was amplified by standard PCR reactions using universal primers (LepF: ATTCAACCAAT-CATAAAGATATTGG, LepR: TAAACTTCTGGATGTCCAAAAAATCA) as in Hajibabaei et al. [45], and a BLAST search was carried out with BioEdit 7.0.5.3 to identify the best-fit mitochondrial contigs [46].

Phylogenetic Analysis
Twenty mitochondrial genomes were employed for phylogenetic analysis.Ten newly sequenced and four GenBank-available chloropid mitochondrial genomes were used as ingroups, covering all four recognized subfamilies (Table 1).Sequences from Syrphidae, Sepsidae, Lauxaniidae, Agromyzidae, and Milichiidae were selected as outgroup taxa (Table 1).
PhyloSuite v1.2.3 was used from mitochondrial genome extraction to matrix preparation [49].Each PCG and rRNA was aligned using the MAFFT module under the '--auto' strategy [53].All ambiguously aligned sites were removed using trimAl [54].Alignments of individual genes were concatenated to build different datasets, and TreeSuite [55] was used to evaluate their saturations (Figures S3-S6).Four datasets and partitioning scheme combinations were prepared for phylogenetic analyses: (1) NTR, consisting of 13 PCGs and 2 rRNAs, partitioned by genes (15 partitions) consisting of 13,200 residues; (2) NT123R, consisting of 13 PCGs and 2 rRNAs, partitioned by genes and codon positions (41 partitions) consisting of 13,200 residues; (3) AA, consisting of amino acid sequences of 13 PCGs, partitioned by genes (13 partitions) consisting of 13,200 residues; (4) DegenR, consisting of 'degenerated' 13 PCGs and 2 rRNAs, partitioned by genes (15 partitions) consisting of 13,200 residues.The degenerated PCGs were generated using the Degen script [56,57], wherein all synonymous sites were reassigned according to the IUPAC ambiguity nomenclature.Phylogenetic trees were constructed under maximum likelihood (ML) methods and Bayesian inference (BI).ML analyses were carried out using IQ-TREE v.2.1.3[58].Datasets were partitioned and model-tested in ModelFinder [59] as implemented in IQ-TREE.We found the best partition scheme after merging possible partitions ('-MFP+MERGE' command) and determining the best scheme under the Bayesian information criterion (BIC).The best-fitting models were used for phylogenetic reconstructions ('-p' command).An initial 1000 parsimony trees were generated in IQ-TREE with the command '-ninit 1000 ′ , and the 100 trees with the fewest steps were used to initialize the candidate set (-ntop 100), considering all possible nearest neighbor interchanges (-allnni).These 100 trees were maintained in the candidate set during the ML tree search (-nbest 100), and unsuccessful runs were terminated after 1000 iterations (-nstop 1000).Perturbation strength was set to 0.2 (-pers 0.2), as recommended for datasets with many short sequences.We applied nearest-neighbor interchange (NNI) branch swapping to improve the tree search and limit overestimating branch supports due to severe model violations ('-bnni' command).Node supports were computed with 1000 UFBoot ('-B' command) replicates [60,61] and SH-aLRT ('-alrt' command) [62].BI analysis was performed using MrBayes 3.2.7 [63].PartitionFinder v2.1.1 was used to assess the optimal partitioning strategy and substitution model using the greedy algorithm and BIC criterion [64].Two independent runs were executed for 1-2 million generations, with sampling occurring every 1000 generations.Additionally, four independent Markov Chain Monte Carlo (MCMC) chains were employed, consisting of three heated chains and a cold chain, and the initial 25% of samples were discarded as burn-in.When the average standard deviation of split frequencies fell below 0.01, we considered that stationarity had been reached.The phylogenetic trees generated in this study were visualized using Figtreev1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 25 November 2018)).

Conclusions
This study presents the first comprehensive comparative analysis of mitochondrial genomes for Chloropidae, providing valuable insights into the phylogeny and evolution of this family.The comparative analysis revealed that Chloropidae have a gene arrangement that is identical to other dipterans.Furthermore, three conserved intergenic sequence blocks were identified (ND1 and tRNA Ser (UCN) , tRNA Glu and tRNA Phe , tRNA His and ND5) in the mitochondrial genomes.Evolutionary rates within Chloropidae vary significantly across subfamilies, with Chloropinae exhibiting higher rates than the other three subfamilies.Moreover, the phylogenetic results supported the monophyly of Chloropidae but failed to construct a well-supported hypothesis regarding the phylogenetic relationships between four subfamilies, possibly due to rapid evolution in grass flies.

Figure 6 .
Figure 6.Phylogenetic trees of Chloropidae based on mitochondrial genome data.(A) Topology from ML inferences based on the NTR dataset showing relationships consistent with those recovered by NTR-BI, NT123R-BI, AA-ML, and DegenR-BI; (B) topology from ML inferences based on NT123R dataset showing relationships consistent with those recovered by DegenR-ML; (C) topology from BI based on the AA dataset.Squares at the nodes represent Bayesian posterior probabilities for 1, 2, 5, and 6 and ML bootstrap values for 3, 4, 7, and 8.The dataset of NTR corresponds to 1, 3 and 2, 4, AA to 5 and 7, DegenR to 6 and 8.A black square indicates posterior probabilities of 1.00 or an ML bootstrap of 100; a grey square indicates posterior probabilities between 0.90 and less than 1.00, or an ML bootstrap between 70 and less than 100; a white square indicates posterior probabilities less than 0.90, or an ML bootstrap less than 70; "ns" indicates a lack of support.Additionally, an asterisk is used to indicate posterior probabilities of 1.00 or ML bootstraps of 100 in all eight trees.

Figure 6 .
Figure 6.Phylogenetic trees of Chloropidae based on mitochondrial genome data.(A) Topology from ML inferences based on the NTR dataset showing relationships consistent with those recovered by NTR-BI, NT123R-BI, AA-ML, and DegenR-BI; (B) topology from ML inferences based on NT123R dataset showing relationships consistent with those recovered by DegenR-ML; (C) topology from BI based on the AA dataset.Squares at the nodes represent Bayesian posterior probabilities for 1, 2, 5, and 6 and ML bootstrap values for 3, 4, 7, and 8.The dataset of NTR corresponds to 1, 3 and 2, 4, AA to 5 and 7, DegenR to 6 and 8.A black square indicates posterior probabilities of 1.00 or an ML bootstrap of 100; a grey square indicates posterior probabilities between 0.90 and less than 1.00, or an ML bootstrap between 70 and less than 100; a white square indicates posterior probabilities less than 0.90, or an ML bootstrap less than 70; "ns" indicates a lack of support.Additionally, an asterisk is used to indicate posterior probabilities of 1.00 or ML bootstraps of 100 in all eight trees.

Funding:
This research was supported by the National Natural Science Foundation of China (32270482), Science & Technology Fundamental Resources Investigation Program (Grant No. 2022FY202100) and the 2115 Talent Development Program of China Agricultural University for Ding Yang and Xuankun Li.

Table 1 .
Information of samples for the phylogenetic analyses used in the study.